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EC number: 204-399-4
CAS number: 120-47-8
Based on Ames Tets on Ethyl paraben and read across from methyl and
Toxic effects of the test item were noted in all tester strains
evaluated in experiment I and II.
In experiment I toxic effects of the test item were observed in
tester strains TA 98 and TA 1537 at a concentration of 5000 µg/plate
(with and without metabolic activation). In tester strain TA 1535 toxic
effects of the test item were noted at concentrations of 2500 µg/plate
and higher (with and without metabolic activation). In tester strains
TA 100 and TA 102 toxic effects of the test item were observed at
concentrations of 2500 µg/plate and higher (without metabolic
activation) and at a concentration of 5000 µg/plate (with metabolic
experiment II toxic effects of the test item were noted in tester
strains TA 98 and TA 102 at concentrations of 2500 µg/plate and higher
(with and without metabolic activation). In tester strains TA 100 and TA
1535 toxic effects of the test item were noted at concentrations of 1000
µg/plate and higher (without metabolic activation) and at concentrations
of 2500 µg/plate and higher (with metabolic activation). In tester
strain TA 1537 toxic effects of the test item were observed at
concentrations of 1000 µg/plate and higher (with and without metabolic
In a reverse gene mutation assay in bacteria, strains TA98, TA100,
TA1535, TA1537 and TA102 of S. typhimurium were exposed to Ethylparaben
at concentrations of 10.0,
31.6, 100, 316, 1000, 2500 and 5000 µg/plate (experiment I) and 3.16,
10.0, 31.6, 100, 316, 1000, 2500 and 5000
µg/plate (experiment II), in the presence and absence of
mammalian metabolic activation according to the plate incorporation
method (experiment I) and the pre-incubation method (experiment II).
Ethylparaben was tested up to the limit concentration of 5000 µg/plate
in all tester strains used.
The positive controls induced the appropriate responses in the
corresponding strains. There was no evidence of induced mutant
colonies over background.
This study is classified as acceptable. This study satisfies the
requirement for Test Guideline OPPTS 870.5100; OECD 471 for in vitro
mutagenicity (bacterial reverse gene mutation) data.
The test item was evaluated for gene mutation test in CHO AA8 cells,
according to OECD TG 476 " In vitro Mammalian cell genen mutation test
using hprt and xprt genes" adopted on 29th july 2016.
The test item was found soluble in DMSO at 200 mg/mL. Slight
precipitation was observed at 2 mg/ml. No change in pH and precipitate
at and up to 2 mg/mL in culure medium. Based on the result of
solubility, pH and precipitation test an initial cytotoxicity test was
conducted at concentrations of 0.0625, 0.125, 0.25, 0.5 1 and 2 mg/mL
using DMSO as vehicle in the presence and absence of metabolic
activation (3 to 6 hours). The result of the initial cytotoxicity test
indicated that the test item was not cytotoxic to CHO AA8 cells, as the
relative survival of the treated CHO AA8 cellsat the test concentrations
up to 2 mg/mL was in a range of 71.91 to 86.21 % when compared with the
respective vehicle control, both in the presence and absence of
metabolic activation. The gene mutation test was conducted at
concentrations of 0.25, 0.5, 1 nd 2 mg/mL in for plates/groups in the
presence and absence of metabolic activation (3 to 6 hours) Benozo(a)
pyrene and 4 Nitroquinoline N-oxide were used as positive controls for
the gene mutation test. Cytotoxicity was assessed by determining the
adjusted cloning efficiency and relative survival in the test.
There was no statistically significant increase in number of mutant
colonies at any of concentrations tested when compared with the vehicle
control. Positive controls resulted in mutant frequencies, which were
statistically significant when compared with the vehicle control.
Based on the results obtained, the test item is considerd as
non-mutagenic and and up to the concentration of 2 mg/mL, both in the
presence and absence of metabolic activation under the tested laboratory
was not continued due to exceedingly severe cytotoxic effects
was performed in two independent experiments, using identical
experimental procedures. In the first experiment the treatment period
was 4 hours with and without metabolic activation. The experimental part
without metabolic activation was prematurely terminated due to microbial
contamination. This part of the experiment was repeated as experiment
IA. The data of experiment IA are reported as experiment I without
metabolic activation. The second experiment was performed with a
treatment time of 4 hours with and 24 hours without metabolic activation.
cytotoxic effects defined as a reduction of the relative cloning
efficiency I and/or relative cell density to values below 50% in both
parallel cultures were noted in the first experiment at 336.0 µg/mL with
and at 93.0 µg/mL without metabolic activation. In the second experiment
cytotoxic effects as described above occurred at 336.0 µg/mL with
metabolic activation. In the experimental part of the second experiment
without metabolic activation a very steep cytotoxic gradient was
observed. Borderline cytotoxicity was noted at 112.0 µg/mL (relative
cell density of 51.9 and 50.4%). Evaluation of any data on mutagenicity
was impossible at the next higher concentration of 168.0 µg/mL as
exceedingly severe cytotoxic effects completely inhibited the cell
growth. The recommended cytotoxic range of approximately 10- 20%
relative cloning efficiency I or relative cell density
with and without metabolic activation.
and reproducible increase in mutant colony numbers/106 cells
was observed in the main experiments up to the maximum concentration.
The mutation frequency remained within the historical range of solvent
controls. The induction factor did not reach or exceed the threshold of
3 time the mutation frequency of the corresponding solvent control.
regression analysis (least squares) was performed to assess a possible
dose dependent increase of mutant frequencies. A significant dose
dependent trend of the mutation frequency indicated by a probability
value of <0.05 was solely detected in the first culture of the first
experiment with metabolic activation. This trend however, was judged as
irrelevant since it actually was reciprocal, going down versus
increasing concentrations.In both experiments of this study (with and
without S9 mix) the range of the solvent controls was from 9.5 up to
22.0 mutants per 106 cells; the range of the groups treated
with the test item was from 0.0 up to 27.3 mutant colonies per 106 cells.
EMS(150 µg/mL) and DMBA (1.1 µg/mL) were used as positive controls and
showed a distinct increase in induced mutant colonies.
TABLE 1. SUMMARY
OF MICRONUCLEI INCIDENCE WITH METABOLIC ACTIVATION FOR 3 TO 6 HOURS
Average % of Cytotoxicity
Total No. of Binucleates
Total No. of Micronucleus
Average Percentage of Micronucleus
TABLE 2. SUMMARY
OF MICRONUCLEI INCIDENCE WITHOUT METABOLIC ACTIVATION FOR 3 TO 6 HOURS
3 SUMMARY OF MICRONUCLEI INCIDENCE WITHOUT METABOLIC ACTIVATION FOR 20
TO 24 HOURS
The test item was evaluated for the formation of micronuclei in the
cytoplasm of interphase cells according to OECD TG 487 “In vitro
Mammalian Cell Micronucleus Test” .Test item was found soluble in
dimethyl sulphoxide at 200 mg/mL. Moderate and mild precipitation was
observed at 2 and 1 mg/mL respectively. No precipitation was observed in
any other concentration tested. No change in pH was observed in any of
the concentrations tested. Hence, 2 mg/mL was selected as highest
concentration for initial cytotoxicity test and other concentrations
tested were 0.125, 0.25, 0.5, 1 and 2 mg/mL using DMSO as a vehicle. The
experiment was conducted with and without metabolic activation. The test
item was assessed in proliferated lymphocytes in duplicates by exposing
for a short term (3 to 6 hours, with and without metabolic activation)
and a long term (20 to 24 hours, without metabolic activation).
Cytokinesis was blocked using Cytochalasin B, the cells were harvested
and slides were prepared. In order to assess the cytotoxicity of the
test item, the Cytokinesis-Block Proliferation Index (CBPI) was
calculated for cultures treated with the test item and vehicle control.
In initial cytotoxicity test, the test item at the concentrations of
0.125, 0.25, 0.50, 1 and 2 mg/mL resulted in a percentage reduction of
average Cytokinesis-Block Proliferation Index (CBPI) was in the range of
1.67 to 30.00 at the tested concentrations in both short term and long
term treatment. The percentage reduction of average Cytokinesis-Block
Proliferation Index (CBPI) was not greater than 45±5% at 2 mg/mL. Hence
2 mg/mL was selected as highest concentration for the micronucleus test.
Other concentrations tested were 0.5 and 1 mg/mL.
In micronucleus test, the test item was tested at the concentrations of
0.5, 1 and 2 mg/mL for short term in presence and absence of metabolic
activation and long term treatment in the absence of metabolic
activation system. The test item did not induced cytotoxicity up to 2
mg/mL when compared to vehicle control. No statistical significant
increase in the percentage of micronuclei in binucleated cells observed
in any of the tested concentration when compared to the vehicle control.
The positive controls resulted increase in the micronuclei frequency
with statistical significance at 95% level of confidence (p<0.05) under
identical conditions, when compared with the vehicle control. This
demonstrated the sensitivity of test system towards positive control and
confirmed that the test conditions were adequate and within the range of
Based on the results obtained, it is concluded that the test item
Propylparaben is non clastogenic and/or non aneugenic in cultured human
lymphocytes at and up to 2 mg/mL both in short term and long term
treatment, both in presence and absence of metabolic activation as it
showed no evidence of increase in the induction of micronuclei under the
TABLE 1. SUMMARY
OF MICRONUCLEI INCIDENCE WITH METABOLIC ACTIVATION FOR 3 TO 6 HOURS for
the read-across substance propyl paraben
3 SUMMARY OF MICRONUCLEI INCIDENCE WITHOUT METABOLIC ACTIVATION FOR 20
TO 24 HOURS
The potential of Methylparaben to induce chromosome aberrations was
investigated in Chinese hamster lung cells in vitro. The test
compound was tested at 125 µg/mL with and without metabolic activation.
Benzo[a]pyrene as reference mutagen showed an increase in the number of
Methylparaben did not induce chromosome aberrations in the absence of S9
mix but was positive in the presence of S9 mix.
Methylparaben is therefore considered to non-mutagenic without and
slightly mutagenic with metabolic activation.
Methylparaben was tested in the dominant lethal assay in rats. The test
item was suspended in 0.85% saline and dosed at 5, 50, 500 and 5000
mg/kg bodyweight to male rats (acute: single dose; subacute: 5 doses at
5 consecutive days), upon the results of the previously conducted dose
range finding study. According to the test procedure the animals were
sequentially mated to 2 females per week for 8 weeks (7 weeks in the
subacute study). Females were killed at 14 days after mating and at
necropsy the uterus was examined for the number of Corpora lutea, early
deaths, late fetal deaths and total implantations.
Triethylene Melamine (TEM) was used as positive control substance and
administered intraperitoneally at a dose of 0.3 mg/kg bodyweight. TEM
caused significant preimplantation loss and embryo resorption during the
first 5 weeks.Saline was used as negative control.
Methylparaben is considered to be non-mutagenic in rats in this dominant
lethal assay when using dosages of 5, 50, 500 and 5000 mg/kg bw/d since
no dose response or time trend patterns, which would suggest an effect,
The in vitro genetic toxicity of ethylparaben was investigated in
a bacterial reverse mutation assay (Ames test) according to OECD 471 and
GLP (Donath, 2012). Two independent experiments (plate incorporation and
preincubation) were conducted with S. typhimurium strains TA 1535, TA
1537, TA 98, TA 100 and TA 102 at concentrations up to 5000 µg/plate
with and without metabolic activation. The test substance did not induce
reversions in any of the strains tested with or without metabolic
activation. The used positive controls were valid. In the plate
incorporation assay, cytotoxicity was observed in tester strains TA 98
and TA 1537 at 5000 µg/plate and in TA 1535, TA 100 and TA 102 at
concentrations of above 2500 µg/plate. After preincubation, cytotoxicity
was noted in tester strains TA 100, TA 1535 and TA 1537 at
concentrations of above 1000 µg/plate and in TA 98 and TA 102 at
concentrations of above 2500 µg/plate. No precipitation of the test
substance was observed in any experiment up to the highest dose tested.
In a further bacterial reverse mutation assay (Ames test) the
genetic toxicity of ethylparaben was determined in tester strains TA
1535, TA 1537, TA 98, TA 100, TA 92 and TA 94 with metabolic activation
(Ishidate, 1984). After preincubation with concentrations up to 5000
µg/plate, the test substance was not genotoxic in any tester strain.
There are no further data available on the genetic toxicity of
ethylparaben. However, there are reliable data for methylparaben and
propylparaben which are structurally related to ethylparaben.Therefore,
read-across was performed based on an analogue approach. For a detailed
justification of the analogue approach, please refer to section 13 of
the technical dossier. The target substance and the source substances
form a homologue series of esters of p-hydroxybenzoic acid and
differonly in the length of the alkyl side chain, which contains 1, 2 or
3 carbon atoms for methylparaben, ethylparaben and propylparaben,
The toxicokinetic behaviour of the target and the source
substances is comparable. The substances were shown to be rapidly
absorbed, metabolised and excreted mainly via urine. The main
metabolites of the three substances were p-hydroxybenzoic acid,
p-hydroxyhippuric acid and their glucuronic acid conjugates.
Available data on ethylparaben did not show reactive properties in
bacterial mutation studies (Ames test) and no genotoxic effects were
observed for propylparaben in several in vitro studies, i.e. Ames test
and HPRT test as well as for methylparaben in vivo. Additionally, no DNA
binding alerts were found for the target and the source substances by
OECD Toolbox profiling. It is considered that there is no evidence for
generation of chemically reactive metabolites for the target and the
In conclusion, as methylparaben, ethylparaben and propylparaben
were shown to have comparable toxicological properties, it is considered
appropriate to read-across from methylparaben and propylparaben to
The induction of gene mutations in mammalian cells by
propylparaben was assessed in a HPRT test according to OECD 476 and GLP
(Wollny, 2012). The test was conducted in two independent experiments in
V79 cells with metabolic activation at concentrations up to 448 µg/mL
for 4 h and without metabolic activation at concentrations up to 224
µg/mL for 4 and 24 h. Cytotoxic effects were observed in both
experiments at concentrations of above 93 µg/mL without metabolic
activation and at concentrations of above 336 µg/mL with metabolic
activation. No genotoxicity was observed up to the highest
concentrations tested. The used positive controls were valid.
Methylparaben was tested in a dominant-lethal test in rats
according to OECD 478 (Litton Bionetics Inc, 1974). Methylparaben was
applied to male rats in doses up to 5000 mg/kg bw/d by oral gavage in a
single treatment and daily on 5 consecutive days. During the post
exposure period (8 week for the single treatment group and 7 weeks for
the subacute group), the male rats were sequentially mated to 2 females
per week. Two weeks after mating, the females were sacrificed and at
necropsy the uteri were examined for corpora lutea, early deaths, late
fetal deaths and number of total implantations. No dose response
relationship or time trend pattern was observed across the groups.
Therefore, methylparaben was considered to be non-mutagenic in rats in
the dominant-lethal assay.
In an in vivo Mammalian Bone Marrow Chromosome Aberration test
performed in male rats according to OECD 475, methylparaben did not
induce chromosome aberrations up to 500 mg/kg bw/d (Litton Bionetics
Justification for selection of genetic toxicity endpoint
Hazard assessment is conducted by means of read-across from
structural analogues. The available studies on in vitro gene mutation in
bacteria, in vitro gene mutation in mammalian cells and in vivo
chromosome aberration were negative. All available studies are adequate
and reliable based on the identified similarities in structure and
intrinsic properties between source and target substance and overall
quality assessment (refer to the endpoint discussion for further
Short description of key information:
Bacterial reverse mutation assay, Ames test (OECD 471): negative
Mammalian Cell Gene Mutation Test, HPRT test (OECD 476): negative
Rodent Dominant Lethal Test (OECD 478): negative
Mammalian Bone Marrow Chromosome Aberration Test (OECD 475): negative
Endpoint Conclusion: No adverse effect observed (negative)
The available data on genetic toxicity of the test substance and
on surrogate substances does not meet the criteria for classification
according to Regulation (EC) 1272/2008 or Directive 67/548/EEC, and is
therefore conclusive but not sufficient for classification.
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.
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